Screening of chicken OATP1B3 interacting proteins and transcriptional expression analysis of their related genes

【Objective】The purpose of the study was to screen and identify chicken organic anion transporting polypeptide 1B3(OATP1B3) interacting proteins, to explore their effects on the formation of green shell color in eggs, and to provide a reference basis for improving the accuracy of selection of green-shelled laying hen populations. 【Method】Eukaryotic expression vector pEGFP-SLCO1B3 and empty vector pEGFP-C1 were used to transfect eggshell gland epithelial cells of green-shelled Chishui black bone hens, respectively. The chicken OATP1B3 interacting proteins were screened by co-immunoprecipitation(Co-IP) coupled with liquid chromatography and tandem mass spectrometry(LC-MS/MS). By GO functional annotation analysis, KEGG signaling pathway enrichment analysis and protein interaction network analysis, OATP1B3 interaction proteins related to the formation of green shell trait in eggs were identified. At the same time, by constructing SLCO1B3 gene silencing and overexpression vectors to transfect eggshell gland epithelial cells respectively, the expression of some OATP1B3 interaction protein genes(FXR1, CTTN, RPL12 and RPS17) in the eggshell gland epithelial cells was detected when SLCO1B3 gene was overexpressed or silenced. And they were categorized into three types of eggshells according to the eggshell color, namely, white shell, light green, and dark green. The correlation between eggshell color △a^** value and the expression of FXR1, CTTN, RPL12 and RPS17 genes was analyzed. 【Result】A total of 37 proteins interacting with chicken OATP1B3 were identified, among which FXR1, CTTN, RPL12 and RPS17were closely related to the green shell trait of eggs. The overexpression of SLCO1B3 gene could extremely significantly down-regulate the expression of FXR1 gene in the epithelial cells of the eggshell glands(P<0.01, the same below), significantly up-regulate the expression of RPL12 gene(P<0.05, the same below), and extremely significantly up-regulate RPS17 gene expression. When SLCO1B3 gene was silenced, the expression of FXR1 and CTTN genes in eggshell gland epithelial cells was extremely significantly up-regulated, and the expression of RPL12 gene was significantly decreased.The relative expression of FXR1 gene in the hypothalamus of white-shelled type Chishui black bone hens was extremely significantly higher than that of green-shelled type Chishui black bone hens, while the relative expression of RPL12 gene and RPS17 gene in the liver was extremely significantly or significantly higher than that of the green-shelled type Chishui black hens. Moreover, the relative expression of FXR1 gene in hypothalamus extremely significantly positively correlated with the value of eggshell color △a^**, and the relative expression of RPL12 gene in eggshell gland and liver was extremely significantly positively correlated with eggshell color △a^** value. 【Conclusion】The 37 chicken OATP1B3 interac-ting proteins identified by screening are mainly localized in the cytoplasm or nucleus, with protein-binding ability and structural molecular activity, and are mainly involved in cellular processes, biological regulation and energy metabolism. There is a regulatory relationship between SLCO1B3 gene and FXR1, CTTN, RPL12 and RPS17 genes in the epithelial cells of chicken shell gland, in which FXR1 and RPL12 genes play an important role in the formation of green shell trait in eggs.